In the last few years, research in the general area of signal transduction has literally exploded with activity. From vision to cell division, protein kinases serve as key components along signal transduction pathways. In addition, there are compelling reasons to believe that specific protein kinase inhibitors could potentially serve as novel cancer chemotherapeutic agents. However, in contrast to our rapidly developing appreciation of signal transduction and the role of protein kinases along these pathways, the creation of specific protein kinase inhibitors (other than the standard alanine-for-serine substitution in active site-directed peptides) has been painfully slow. This will be partially alleviated by the recent acquisition of the 3- dimensional structure of the cAMP-dependent protein kinase. Nevertheless, while additional crystallographic studies on other protein kinases will undoubtedly be equally beneficial, such research is often fraught with difficulties. In contrast, we have developed a methodology that will allow us to survey the active site structure and specificity of protein kinases in a rapid fashion. In addition, the strategy that we have developed will enable us to acquire data that highlights both the similarities and differences among these family members in the context of inhibitor design. In the work described herein, we will analyze seven separate protein kinases. Simultaneously, we plan to investigate the active site substrate specificity of mutant forms of the cAMP-dependent protein kinase that contain amino acid substitutions present in other protein kinases. This will not only allow us to assess the role of individual active site amino acid residues in controlling substrate specificity, but in addition it should provide a basis for the comparison of protein kinases in terms of the 3-dimensional structure of the cAMP- dependent protein kinase. As the substrate specificity and the factors which control this specificity in individual kinases begins to unfold, we will execute a comprehensive evaluation of a range of protein kinase inhibitors, with the goal of identifying inhibitory functionality that are specific to a particular subset of kinases. Finally, the results of these studies will be integrated to create nonpeptidic protein kinase substrates and inhibitors, species that should be especially useful for the in vivo evaluation of protein kinase action.